Influence of the autotaxin-lysophosphatidic acid axis on cellular function and cytokine expression in different breast cancer cell lines

Previous studies provide high evidence that autotaxin (ATX)-lysophosphatidic acid (LPA) signaling through LPA receptors (LPAR) plays an important role in breast cancer initiation, progression, and invasion. However, its specific role in different breast cancer cell lines remains to be fully elucidated to offer improvements in targeted therapies. Within this study, we analyzed in vitro the effect of LPA 18:1 and the LPAR1, LPAR3 (and LPAR2) inhibitor Ki16425 on cellular functions of different human breast cancer cell lines (MDA-MB-231, MDA-MB-468, MCF-7, BT-474, SKBR-3) and the human breast epithelial cell line MCF-10A, as well as Interleukin 8 (IL-8), Interleukin 6 (IL-6) and tumor necrosis factor (TNF)-alpha cytokine secretion after LPA-incubation. ATX-LPA signaling showed a dose-dependent stimulatory effect especially on cellular functions of triple-negative and luminal A breast cancer cell lines. Ki16425 inhibited the LPA-induced stimulation of triple-negative breast cancer and luminal A cell lines in variable intensity depending on the functional assay, indicating the interplay of different LPAR in those assays. IL-8, IL-6 and TNF-alpha secretion was induced by LPA in MDA-MB-468 cells. This study provides further evidence about the role of the ATX-LPA axis in different breast cancer cell lines and might contribute to identify subtypes suitable for a future targeted therapy of the ATX-LPA axis.

Experimental groups. For measuring the effect of Oleoyl-LPA (LPA 18:1, 857130P; Avanti Polar Lipids Inc., Alabaster, AL, USA) and the LPA receptor 1, 3 (and 2) blocker Ki16425 (Sigma Aldrich) on cell properties, different experimental groups were performed summarized in Table 1. LPA was dissolved according to manufacturer's instructions. Cells were seeded in the corresponding medium supplemented with either 0.1 μM LPA 18:1 or 1 μM LPA 18:1. 2 μM or 20 μM Ki16425 was added one minute before treatment with LPA. For all experimental groups including the control group and in all assays 10% FBS in culture medium was replaced by 0.2% fatty acid free bovine serum albumin (BSA; Sigma Aldrich).
Proliferation assay. MDA-MB-231 cells were seeded at a density of 1 × 10 3 cells/well, MDA-MB-468, MCF-7, BT-474, SKBR-3 and MCF-10A were seeded at a density of 5 × 10 3 cells/well in 96-well plates in triplicate. Every 24 h, the medium was replaced according to the experimental groups, thus, including fresh LPA (Table 1). After an incubation period of 24 h, 48 h, and 72 h, cell proliferation was measured by fluorometric quantification of DNA using CyQUANT Direct Cell Proliferation Assay Kit (Invitrogen/Thermo Fisher, Carlsbad, CA, USA) according to the manufacturer's instructions. 100 μl of 2X detection reagent was added to each well. After the incubation period of 60 min at 37 °C and 5% CO 2 , fluorescence was measured at 480/535 nm (NOVOstar, BMG LABTECH, Ortenberg, Germany). The assay was performed three times for all cell lines.
Migration. For analysis of cell migration, the Oris Assembly Kit (Platypus Technologies, Madison, WI, USA) was used. Cell seeding stoppers were inserted into the wells of a 96-well plate for creating a cell-free area. 5 × 10 4 MDA-MB-231, 7.5 × 10 4 MDA-MB-468, 7.5 × 10 4 MCF-7, 1 × 10 5 SKBR-3, 1 × 10 5 BT-474, or 1 × 10 5 MCF-10A Invasion and transmigration assay. For invasion and transmigration assays, transwell inserts with a pore size of 8 μm were used (ThinCert, Greiner Bio-One GmbH, Frickenhausen, Germany). The transwells for invasion assays were coated with 2.4 mg/ml collagen type I from bovine skin (Sigma-Aldrich). The transwells were placed into a 24-well plate and seeded in technical triplicate with 1 × 10 5 cells in 300 μl of the corresponding control medium. The lower chamber was filled with medium according to the experimental groups (Table 1). After 8 h at 37 °C and 5% CO 2 , the transwell inserts were washed with PBS, fixed with ice-cold methanol and stained with 1 μg/ml 4′,6-diamidino-2-phenylindole (DAPI) for 10 min (Life technologies, Carlsbad, CA, USA). Non-migrated or non-invaded cells in the inner part of the transwells were removed by a PBS-coated cotton swab wiped in twisting motions. Transmigrated and invaded cells were counted in the four quadrants of each transwell in 40-fold magnification (Olympus IX83, cellSens Software). The assay was performed two times for all cell lines (control was set 1).
To investigate the influence of the LPA concentration on the transmigration rate of MDA-MB-231 cells, a seven point dose response curve was performed. Therefore, transmigration assay was conducted as described above, whereby 0 µM (control), 0.05 µM, 0.1 µM, 0.5 µM, 1 µM, 5 µM, and 10 µM LPA were added to the control medium in the lower chamber. This assay was performed in technical triplicate and in two replicate experiments.
Real-time qPCR. For analysis of LPAR1-3 expression of different cell lines, RNA was extracted with the RNeasy Mini Kit (Qiagen, Hilden, Germany) and reverse transcribed into cDNA by using the QuantiTect Reverse Transcription Kit with a DNase I incubation (Qiagen). Quantitative real-time PCR was performed with the SsoAdvanced Universal SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA) in a Light Cycler (Bio-Rad CFX96). All kits were used according to the manufacturers' recommendation. Detected transcript levels were normalized to the four different housekeeping genes Coiled-Coil Serine Rich Protein 2 (CCSER2), Symplekin Scaffold Protein (SYMPK), Ankyrin Repeat Domain 17 (ANKRD17), and Pumilio RNA Binding Family Member 1 (PUM1) using the 2 −ΔCT -method. Primers were selected according to Tilli et al. 23 and are summarized in Table 2. The assay was performed in technical triplicate and in three replicate experiments.
Enzyme-linked immunosorbent assay (ELISA) measurements. 6  , respectively, were seeded in T25 cell culture flasks in their standard culture medium. When reaching 80% confluency, the different breast cancer cell lines were stimulated with 1.0 μM LPA in culture medium containing 0.2% fatty acid free BSA. After 24 h, supernatants from all cell lines were collected and levels of secreted IL-8 were detected by highly sensitive IL-8 ELISA Kit (IL-8 Human ELISA Kit; Invitrogen, Waltham, MA, USA) according to the manufacturer's instructions using the NOVOstar. Levels of secreted IL-6 and TNF-alpha were detected by using IMMULITE 1000 Immunoassay System (Siemens, Germany). According to the standard instructions, 100 μl of cell culture supernatants for IL-6 and TNF-alpha and 50 μl of cell culture supernatants for IL-8 measurements was used. The assay was performed in three replicate experiments.

Statistics.
For cell functional assays, differences between groups were analyzed using the Kruskal-Wallis test, followed by Dunn's test for post-hoc analysis (GraphPad Prism version 8.3.0 for Windows; La Jolla, CA, USA). ELISA assays were analyzed using the Mann-Whitney U test; the asymptotic significance was used (SPSS v.21.0 Software/IBM, Armonk, NY, USA). Error bars in the graphs indicate the standard deviations (SD). A p-value ≤ 0.05 was considered significant.   (Table 3, Fig. 1). High concentrated Ki16425 trended to inhibit this stimulatory effect (data of LPA 0.1 μM ± Ki16425 2 μM/20 μM not shown). Further, there was a stimulatory trend (= statistically non-significant) of LPA in MCF-7 cells. In contrary, LPA did not stimulate cell proliferation of BT-474, SKBR-3, and MCF-10A cells (Table 3). Ki16425 alone had no significant effect on cell proliferation ( Supplementary Fig. S1).  Table 4). High-concentrated Ki16425 inhibited these stimulatory effects significantly in MDA-MB-231 and slightly reduced stimulation in MCF-7 and SKBR-3 cells (data of LPA 0.1 μM ± Ki16425 2 μM/20 μM not shown). The sole addition of high-concentrated Ki16425 had no effect on migration rates of       www.nature.com/scientificreports/

Discussion
The role of ATX-LPA signaling in malignancies and its involvement in tumor progression has been repeatedly shown in multiple studies so that the ATX-LPA axis evolved to a potential target of tumor therapies [24][25][26] . Breast cancer with its numerous subtypes represents one of the potential examples of targeted therapy. Despite extensive evidence that LPA is associated with tumorigenesis of breast cancer, a dedicated subtype analysis including different breast cancer cell lines and functional assays has not yet been performed. Previous studies have mostly examined the influence of the ATX-LPA axis on single cell functions of one or two subtypes. Thereby, various experimental designs lead to partially contradicting results 27,28 . Different experimental setups, including e. g. different incubation times, materials, and LPA concentrations, could lead to various results and impede the comparison of different cell lines among different studies. Therefore, the aim of the study was to elucidate the role of ATX-LPA signaling in different breast cancer subtypes within one single study. We analyzed the influence of the ATX-LPA axis on functional properties and IL-8, IL-6 and TNF-alpha secretion of different breast cancer cell lines categorized into five subtypes according to Dai et al. 29 :   www.nature.com/scientificreports/ The LPA-induced cell responses varied among the investigated cell lines. Whereas MDA-MB-231, MDA-MB-468, MCF-7, and to a certain degree also SKBR-3 were stimulated dose-dependently by LPA, BT-474 and MCF-10A reacted to LPA with non-stimulated or even inhibited functional properties. The effect of LPA is mediated by signals conducted by at least six specific G protein-coupled LPAR1-6. The reason for the described different reactions of the cell lines to LPA is most likely to be found in their distinct expressions of LPARs. Multiple studies demonstrated that expression profiles vary among tumor types and subtypes 18,30 . LPAR1-3 play a crucial role in breast cancer (reviewed by 4 ). Therefore, we used the LPAR1, LPAR3 and weak LPAR2 antagonist Ki16425 5,16 to test if the above-described LPA-induced reactions were signaled mainly via LPAR1, LPAR3 (and LPAR2).
LPA stimulated significantly proliferation and migration of MDA-MB-231 cells. Further, our results showed a dose-dependent inhibition of the LPA-stimulated responses by Ki16425. Whereas previous studies found that TNBC cell lines like MDA-MB-231 express the highest levels of LPAR3 within several breast cancer subtypes 14,15 , expression of LPAR3 by MDA-MB-231 could not be detected in other studies 18,31 . Further studies clearly demonstrated that MDA-MB-231 mainly expressed LPAR1 compared to LPAR2 and LPAR3 27,[30][31][32][33] . This is in line with the RT-PCR results in the present study. MDA-MB-231 cells expressed very high levels of LPAR1, lower levels of LPAR3 and hardly LPAR2. This strongly supports the involvement of LPAR1 in LPA-induced proliferation and migration and underlines previous assumptions of a major role of LPAR1 in MDA-MB-231 cells 34 . In accordance with our migration results, dose-response studies showed that a higher dose of 1 μM LPA was associated with increased migration rates of MDA-MB-231 cells 35 . However, from a certain level of LPA concentration, a decrease in the stimulatory effect was reported, so that dose response experiments follow a bell-shaped curve 30,36 . In the present study, high concentrated LPA (1 µM) led to a higher increase in cell migration and proliferation compared to the lower concentration (0.1 µM), whereas the lower concentration of LPA evoked higher cell invasion and transmigration rates compared to the higher concentration. This corresponds with the findings of Chen et al., who observed higher invasion rates of MDA-MB-231 in 0.1 μM LPA compared to 1 μM LPA 27 . Earlier studies reported a decisive role of LPAR1 in invasion and transmigration 30,33 and it was emphasized that efficiency in transducing signals differs between LPARs with ligand-dependent endocytosis of LPAR1 at higher concentrations of LPA 27,37 . Assuming that LPA binds primarily to LPAR1 in MDA-MB-231 cells signaling invasion and transmigration, and regarding the transmigration dose-response experiment in the present study (Supplementary Fig. S2), our observations support this theory: At 1.0 μM or higher concentrations of LPA, LPAR1 might be more rapidly endocytosed so that 1.0 μM has only a minor stimulatory effect, which cannot be sufficiently inhibited by Ki16425. Chen et al. suggested a possible cooperation of distinct LPAR. Further, efficacy of distinct LPAR seems to vary depending on the LPA concentration with LPAR2 operating predominantly at higher levels of LPA. Migration and proliferation might signal via multiple LPAR and thus, show a greater response at higher levels of LPA. Hopkins et al. have already suggested the involvement of multiple receptors in LPA-induced proliferation in MDA-MB-231 cells 34 . Additionally, similar to our results, they showed inhibition of proliferation by increasing concentrations of Ki16425.
Proliferation of MDA-MB-468 cells was significantly stimulated by LPA, while there was a lower effect on cell invasion and transmigration. In our experimental setting, the MDA-MB-468 cells expressed highest levels of LPAR3 and lower levels of LPAR2. This indicates that those receptors are also involved in signaling the induced effects. LPAR3 might thereby occupy a greater role in proliferation than in invasion and transmigration. Ki16425 mostly did not show a complete inhibition of the stimulatory effect so that our results suggest the involvement of several LPAR in mediating LPA responses. Within the investigated cell lines, MCF-7 showed a high expression level of LPAR2, which is also consistent with existing data 18,27,34 . LPA showed a stimulatory migratory effect in MCF-7 cells in the present study. Previous studies described both LPA-stimulated 28 and LPA-inhibited invasion of MCF-7 cells 27 . Those differences might occur due to varying experimental setups, such as different incubation times or transwell coatings, or the duration of serum starvation. Whereas a significant enhancement in migration rates of MCF-7 cells was observed, there was a lesser effect on transmigration rates. Varying results of invasion/ transmigration and migration might be explained by acting through different LPAR and underlying molecular pathways. In invasion/transmigration assays, LPA was added to the bottom chamber and cells in the transwell system were allowed to invade and migrate in response to a chemoattractant gradient. In contrary, the migration assay did not provide migration via chemotaxis so that differences between these assays are conceivable.
In the present study, LPAR1-3 levels of the HER2-positive SKBR-3 cells were very low and no stimulatory effect of LPA could be observed in proliferation, invasion, and transmigration assays. Previous studies described very low LPAR1 and LPAR3 expression of HER2-positive cell lines and no stimulatory effect of LPA in chemotaxis assays 18,27 . Nevertheless, the present data shows a significant LPA-induced enhancement of migration rates after 72 h in SKBR-3 cells. However, this stimulation could not be sufficiently inhibited by Ki16425. Distinct/further LPAR that are not inhibited by Ki16425 might be a reason for this observation and should be investigated in further studies. Moreover, higher concentrations of LPA might be necessary to induce a stimulatory effect. Within this study, BT-474 and MCF-10A cells reacted to LPA with non-stimulated or even inhibited transmigration and invasion comparable to previous studies 18 . Stable expressions of LPAR1 in MCF-10A cells and the acquisition of an invasive phenotype of LPA-stimulated MCF-10A cells was reported in three-dimensional experiments, nevertheless, no increasing migration rates could be observed towards LPA 31 .
ATX-LPA signaling was previously shown not only to influence cellular function, but also to activate an inflammatory cycle in breast cancer cells 19 . It was repeatedly shown that LPA crosstalks, i.a., with cytokine receptors 38 . Thereby, regulation of IL-8 and IL-6 by LPA mainly in ovarian cancer, but also in some breast cancer cell lines was described [39][40][41][42] . The upregulation of IL-8 is associated with breast cancer progression 43 and the activation of breast stromal adipocytes 44 . Besides its tumor-promoting role including the proliferation, migration, and survival of cancer cells, IL-8 was also attributed a role in extracellular matrix remodeling, such as enhancing the immunosuppressive microenvironment and promoting epithelial-to-mesenchymal transition www.nature.com/scientificreports/ (EMT) 43,45,46 . IL-6 signals tumor proliferation, angiogenesis, stromal cell activation and immunomodulation of the microenvironment and might induce drug resistance [47][48][49] . Previous investigations reported a LPA-enhanced IL-8 production by TNBCs and MCF-7 39,41,50 , and no/slight stimulation of IL-8 production in SKBR-3 cells 40 . We therefore investigated the effect of 1.0 μM LPA on the secretion of IL-8 among the different breast (cancer) cell lines. In the present study, LPA stimulated the secretion of IL-8 in MDA-MB-468. In contrast, LPA did not enhance the secretion of IL-8 in the other investigated cell lines. LPAR2 was regarded as important receptor for signaling cytokine secretion 6 51 . On the other hand, TNF-alpha is an important pro-inflammatory cytokine in the tumor microenvironment and is secreted both by breast cancer and by surrounding stromal cells. It is attributed a role in tumor proliferation, tumorangiogenesis and EMT in breast cancer 51 . Further, TNF-alpha enhances ATX production in the adjacent adipose tissue 19 . A recent study showed that LPA induces secretion of TNF-alpha in ovarian cancer cells and thus, adjusts an inflammatory network in ovarian cancer 52 . However, to our knowledge, no prior study has revealed an LPAinduced TNF-alpha secretion in breast cancer cells. Whereas we observed an enhanced release of TNF-alpha in MDA-MB-468 cells, we found a decreased secretion of TNF-alpha in MDA-MB-231 cells after incubation with LPA. Wang et al. established the involvement of LPAR2 in the mediation of TNF-alpha induction and reported a minor or even inhibitory role of LPAR3 in TNF-alpha release in ovarian cancer cell lines 52 . LPA-induced secretion of TNF-alpha in MDA-MB-468 and the corresponding LPAR expression profile of this cell line in the present study support the proposed involvement of LPAR2 in TNF-alpha production. Since LPAR3 is most highly expressed in MDA-MB-468, the reported potential inhibitory effect of LPAR3 does not appear to be strong enough to suppress LPAR2-mediated stimulation completely. The inhibition of TNF-alpha in MDA-MB-231 cells might indicate an inhibitory function of LPAR1. However, further and more detailed cytokine analyses are necessary to elucidate the underlying pathways.
The described differences in LPA-mediated cellular responses and IL-8, IL-6 and TNF-alpha expression among the breast (cancer) cells underline previous findings that ATX-LPA-LPAR signaling is a complex issue, as it depends on the interplay on various factors, such as expression of LPAR, LPA concentration, and the same LPAR showing tumor-promoting as well as anti-tumorigenic effects. Several underlying downstream signaling pathways have already been identified 53,54 . Nevertheless, further research including further LPAR inhibitors and providing mechanistic insights into underlying molecular pathways is needed to fully elucidate the complex interactions. Additionally, the role of LPA turnover by lipid phosphate phophatases (LPPs) in different cell lines and the role of the tumor microenvironment should not be unattended in this issue and be included in further investigations.

Conclusions
Among the examined breast cancer subtypes, LPA dose-dependently stimulated tumor-promoting cellular functions of triple-negative A, triple-negative B, luminal A, and to a certain degree also HER-2 positive subtypes, whereas the luminal B subtype and the non-tumorigenic epithelial cell line were not stimulated by LPA. Depending on the functional assay, Ki16425 inhibited the LPA-induced stimulation in triple-negative breast cancer and luminal A cell lines in a variable intensity and enhanced secretion of IL-8, IL-6 and TNF-alpha in MDA-MB-468 cells. This indicates the interplay of different LPAR in the performed assays. The present study further elucidates the role of the ATX-LPA axis in breast cancer and might contribute to identify suitable subtypes for a promising targeted therapy.

Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.